Analysis of ribosomal RNA (rRNA) genes is one of the most important methods for characterizing the organisms present in an environment, including those associated with medical conditions. The first step of these analyses is usually to apply the polymerase chain reaction (PCR) to specifically amplify the genes of interest. This requires the use of primers that are specific to the rRNA genes, yet capable of accommodating all of the natural variation of the genes. The investigators will analyze sequence variation in rRNA gene primer-binding sites in the currently available data with the goal of improving current primer designs to recognize the full range of relevant organisms. They will also explore trade-offs in using sequence degeneracy, noncanonical nucleotides, and primer annealing stringency to accommodate the observed sequence diversity. In so doing, they will explore the usefulness of strategies that might guide amplifications toward a common template and primer sequence. These goals will use computer analysis of sequences from the databases and experimental comparisons of alternative primer designs and amplification protocols. The results of much of the proposed work will be applicable for molecular analyses based on molecules other than rRNA genes. PUBLIC HEALTH RELEVANCE Microbes play important roles in human health and disease. There are increasing numbers of studies of the mixture of microbes associated with both normal and abnormal conditions. Most in-depth studies are based upon the analysis of particular molecules, most commonly the ribosomal RNAs. The goal of the proposed studies is to improve these methods in the detection of the full diversity of organisms present in the samples. This work will be integrated with on-going analyses of vaginal microbiota in humans.